Multivalent pneumococcal polysaccharide-protein conjugate composition

Abstract

An immunogenic composition having 13 distinct polysaccharide-protein conjugates and optionally, an aluminum-based adjuvant, is described. Each conjugate contains a capsular polysaccharide prepared from a different serotype of Streptococcus pneumoniae (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) conjugated to a carrier protein. The immunogenic composition, formulated as a vaccine, increases coverage against pneumococcal disease in infants and young children globally, and provides coverage for serotypes 6A and 19A that is not dependent on the limitations of serogroup cross-protection. Also described is a method for making an immunogenic conjugate comprising Streptococcus pneumoniae serotype 1 polysaccharide covalently linked to a carrier protein, the method including partial de-O-acetylation of the polysaccharide by mild hydrolysis in an alkaline pH buffer.

What is claimed is: 
     
       1. A method for making an immunogenic conjugate comprising  Streptococcus pneumoniae  serotype 1 polysaccharide covalently linked to a carrier protein, the method comprising:
 (a) reacting purified serotype 1 polysaccharide with an alkaline pH buffer resulting in a partially de-O-acetylated serotype 1 polysaccharide; 
 (b) reacting the partially de-O-acetylated serotype 1 polysaccharide with a mild acid resulting in a neutralized, partially de-O-acetylated serotype 1 polysaccharide; 
 (c) reacting the neutralized, partially de-O-acetylated serotype 1 polysaccharide with an oxidizing agent resulting in an activated serotype 1 polysaccharide; 
 (d) compounding the activated serotype 1 polysaccharide with a carrier protein; 
 (e) reacting the compounded, activated serotype 1 polysaccharide and carrier protein with a reducing agent resulting in a serotype 1 polysaccharide:carrier protein conjugate; and 
 (f) capping unreacted aldehydes in the serotype 1 polysaccharide:carrier protein conjugate resulting in an immunogenic conjugate comprising  Streptococcus pneumoniae  serotype 1 polysaccharide covalently linked to a carrier protein. 
 
     
     
       2. The method of  claim 1 , further comprising purifying the activated serotype 1 polysaccharide prior to compounding with the carrier protein. 
     
     
       3. The method of  claim 1 , further comprising co-lyophilizing the compounded, activated serotype 1 polysaccharide and carrier protein prior to reacting with the reducing agent. 
     
     
       4. The method of  claim 1 , further comprising purifying the immunogenic conjugate. 
     
     
       5. The method of  claim 1 , wherein the alkaline pH buffer is a bicarbonate/carbonate buffer. 
     
     
       6. The method of  claim 1 , wherein the mild acid is acetic acid. 
     
     
       7. The method of  claim 1 , wherein the mild acid is a low ionic strength mineral acid. 
     
     
       8. The method of  claim 7 , wherein the low ionic strength mineral acid is hydrochloric acid. 
     
     
       9. The method of  claim 1 , wherein the oxidizing agent is sodium periodate. 
     
     
       10. The method of  claim 1 , wherein the carrier protein is CRM 197 . 
     
     
       11. The method of  claim 1 , wherein the reducing agent is sodium cyanoborohydride. 
     
     
       12. The method of  claim 1 , wherein capping unreacted aldehydes comprises reacting the serotype 1 polysaccharide:carrier protein conjugate with sodium borohydride. 
     
     
       13. A method for making an immunogenic conjugate comprising  Streptococcus pneumoniae  serotype 1 polysaccharide covalently linked to a carrier protein, the method comprising:
 (a) reacting purified serotype 1 polysaccharide with a bicarbonate/carbonate buffer resulting in a partially de-O-acetylated serotype 1 polysaccharide; 
 (b) reacting the partially de-O-acetylated serotype 1 polysaccharide with acetic acid resulting in a neutralized, partially de-O-acetylated serotype 1 polysaccharide; 
 (c) reacting the neutralized, partially de-O-acetylated serotype 1 polysaccharide with sodium periodate resulting in an activated serotype 1 polysaccharide; 
 (d) purifying the activated serotype 1 polysaccharide; 
 (e) compounding the activated serotype 1 polysaccharide with a carrier protein; 
 (f) co-lyophilizing the compounded, activated serotype 1 polysaccharide and carrier protein; 
 (g) reacting the co-lyophilized, activated serotype 1 polysaccharide and carrier protein with sodium cyanoborohydride resulting in a serotype 1 polysaccharide:carrier protein conjugate; and 
 (h) capping unreacted aldehydes in the serotype 1 polysaccharide:carrier protein conjugate with sodium borohydride resulting in an immunogenic conjugate comprising  Streptococcus pneumoniae  serotype 1 polysaccharide covalently linked to a carrier protein. 
 
     
     
       14. The method of  claim 13 , further comprising purifying the immunogenic conjugate. 
     
     
       15. The method of  claim 13 , wherein the carrier protein is CRM 197 . 
     
     
       16. A method for making an activated  Streptococcus pneumoniae  serotype 1 polysaccharide, the method comprising:
 (a) reacting purified serotype 1 polysaccharide with an alkaline pH buffer resulting in a partially de-O-acetylated serotype 1 polysaccharide; 
 (b) reacting the partially de-O-acetylated serotype 1 polysaccharide with a mild acid resulting in a neutralized, partially de-O-acetylated serotype 1 polysaccharide; and 
 (c) reacting the neutralized, partially de-O-acetylated serotype 1 polysaccharide with an oxidizing agent resulting in an activated serotype 1 polysaccharide. 
 
     
     
       17. The method of  claim 16 , further comprising purifying the activated serotype 1 polysaccharide following oxidation. 
     
     
       18. The method of  claim 16 , wherein the alkaline pH buffer is a bicarbonate/carbonate buffer. 
     
     
       19. The method of  claim 16 , wherein the mild acid is acetic acid. 
     
     
       20. The method of  claim 16 , wherein the oxidizing agent is sodium periodate.